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Membrane androgen receptor ZIP9 induces croaker ovarian cell apoptosis via stimulatory G protein alpha subunit and MAPkinase signaling. Converse A et al. (2017) Recent studies show that androgen-induced apoptosis in Atlantic croaker primary granulosa/theca (G/T) cells and in human breast and prostate cancer cell lines is mediated by the novel membrane androgen receptor ZIP9 which belongs to the SLC39A zinc transporter family. However, the apoptotic signaling pathways remain unclear because ZIP9 activates an inhibitory G protein in human cancer cells, whereas recombinant croaker ZIP9 activates a stimulatory G protein (Gs) in transfected cancer cells. Here we investigated androgen-dependent apoptotic pathways in order to identify the signaling pathways regulated through wild-type croaker ZIP9 in ovarian follicle cells. We show that the ZIP9-mediated apoptotic signaling pathway in croaker G/T cells shares several proapoptotic members with those in human cancer cells, but is activated through a Gsα subunit-dependent pathway. Testosterone treatment of croaker G/T cells increased intracellular zinc levels, Erk activity, caspase 3 activity, mRNA levels of proapoptotic members Bax, p53, and JNK, and the incidence of apoptosis, similar to findings in mammalian cancer cells, but also increased cAMP concentrations. Transfection with small interfering RNA targeting croaker ZIP9 blocked the testosterone-induced increase in bax, p53, and jnk expression. Testosterone-induced apoptosis and caspase 3 activation were dependent on the presence of extracellular zinc and were effectively blocked with co-treatment of inhibitors of the Gsα subunit, adenylyl cyclase, PKA, and Erk activation. These results indicate that ZIP9-mediated testosterone signaling in croaker G/T cells involves multiple pathways, some of which differ from those activated through ZIP9 in human cancer cells even though a similar apoptotic response is observed.//////////////////
Identification and characterization of membrane androgen receptors in the ZIP9 zinc transporter subfamily: I. Discovery in female Atlantic croaker and evidence ZIP9 mediates testosterone-induced apoptosis of ovarian follicle cells. Berg AH 2014 et al.
Rapid, cell surface-initiated, pregenomic androgen actions have been described in various vertebrate cells, but the receptors mediating these actions remain unidentified. We report here cloning and expression of a cDNA from Atlantic croaker (Micropogonias undulatus) ovaries encoding a 33 kDa, 7-transmembrane protein with binding and signaling characteristics of a membrane androgen receptor (mAR) that is unrelated to any previously described steroid receptor. Instead croaker mAR has 81-93% amino acid sequence identity with zinc transporter ZIP9 (SLC39A9) subfamily members, indicating it is a ZIP9 protein. Croaker ZIP9 is expressed in gonadal tissues and in brain, and is upregulated in the ovary by reproductive hormones. ZIP9 protein is localized to plasma membranes of croaker granulosa cells and human breast cancer (SKBR-3) cells stably transfected with ZIP9. Recombinant croaker ZIP9 has a high affinity (Kd 12.7 nM), limited capacity (Bmax 2.8nM/mg protein), displaceable, single binding site specific for androgens, characteristic of steroid receptors. Testosterone activates a stimulatory G protein coupled to ZIP9, resulting in increased cAMP production. Testosterone promotes serum starvation-induced cell death and apoptosis in transfected cells and in croaker ovarian follicle cells that is associated with rapid increases in intracellular free zinc concentrations, suggesting an involvement of zinc in this nonclassical androgen action to promote apoptosis. These responses to testosterone are abrogated by treatment with ZIP9 siRNA. The results provide the first evidence that zinc transporter proteins can function as specific steroid membrane receptors and indicate a previously unrecognized signaling pathway mediated by steroid receptors involving alterations in intracellular zinc.
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Oocyte-cumulus cell interactions regulate free intracellular zinc in mouse oocytes. Lisle RS et al. Zinc increases in the oocyte during maturation and is required for progression and completion of meiosis. The objective of this study was to determine whether cumulus cells regulate the levels of free intracellular zinc in the oocyte during maturation. In the cumulus-oocyte complex (COC), the relative level of free intracellular zinc was almost 4-fold higher in cumulus cells compared to the resident germinal vesicle (GV) stage oocyte. Removal of cumulus cells caused a 4-fold increase in intracellular zinc in the oocyte by one hour after cumulus cell removal, but subsequent co-culture of denuded oocytes with COC decreased free intracellular zinc in the oocyte by 65%. Thus, cumulus cells suppress free intracellular zinc in the oocyte. The zinc transporter proteins Zip6, Zip8, Zip9, Zip10, Zip12, Znt2, Znt4, Znt5 and Znt8 mRNA was higher in oocytes, while Zip1, Zip7, Zip13, Zip14, Znt6, Znt7 and Znt9 mRNA was higher in cumulus cells. Thus a complex zinc transport network is present in the COC. Pretreatment with EGF for 4 hours abolished the ability of cumulus-oocyte complexes to restrict free intracellular zinc in denuded oocytes. Co-culture of denuded MII oocytes with COC lowers free intracellular zinc in mature oocytes. Oocytes matured in vivo or oocytes from older mice had lower levels of free intracellular zinc than oocytes matured in vitro or from younger mice. Thus, a precise mechanism for regulating oocyte zinc homeostasis has been uncovered in the cumulus-oocyte complex that is disrupted with increasing age or by removal of cumulus cells.
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